ELISpot including its variant FLUOROSpot remains the mainstay to measure true secretion of single cells and to assess cellular function (e.g., immunity) in the pre-clinical and clinical settings. However, because it measures only one or two proteins per single cell and provides minimal biological information, it has limited utility for measuring cell biology and physiological functions at the systems level. Because a subset of the cells capable of producing multiple cytokines (up to 40), called polyfunctional cells, dominate the overall response but currently no commercial tools are available to quantify polyfunctional cells at the highly multiplexed level, representing a major barrier in both academic research and pharmaceutical industry to accurately quantify cellular immune responses. Thus, it is necessary to develop a technology that can measure an array of proteins from single cells, and will help address a host of important biological and biomedical questions ranging from immune cell function heterogeneity, stem cell differentiation, drug resistance, angiogenesis, to cell-cell communication networks. Also, this technology is of urgent need in biotech and pharmaceutical industry. To address these challenges, this technology needs to meet all three of the following requirements: (i) Single cell sensitivity, (ii) High multiplicity, and (iii) High content. IsoPlexi has a prototype hand-held technology that for the first time provides the ability to measure many (up to 42) secreted effector proteins at the single T-cell level. At the same time, this device in its envisioned form will be far less costly than existing single-cell instruments, representing a significant market advantage for wide-spread use in pre-clinical and clinical uses. When applied to, but not limited in, the immunotherapeutics space, our technology has the capability to (1) detect the broadest mechanism of action at the single immune cell level, and (2) quantify the effector function of activated T-cell response including polyfunctionality at a broad cellular leve to detect both effective response and potential toxicity (autoimmunity). With Phase 1 support, IsoPlexis plans to conduct the initial industrial R&D to turn this academic technology to a commercial product. We propose to: (1) develop a fully packaged single-cell protein secretion assay device to meet minimum commercial usability and validity standards; (2) develop computational pipelines and a software suite for automated data quantification and informatics. We expect to develop a user-friendly hardware and a software suite for reliable single cell multiplex protein secretion profiling and rapid data analysis. This technology can not only provide comprehensive single cell information to address the biological and biomedical questions in academic research, but also offers a solution for biotech and pharmaceutical industry to evaluate drug/treatment responses at the single cell level.